Best practices for custom sequencing primer use on the NextSeq 1000/2000

Custom sequencing primers are enabled with NextSeq 1000/2000 Control Software v1.2+.

Refer to the NextSeq 1000/2000 custom primers guide for guidance on preparing and adding custom primers to the reagent cartridge, including volumes and concentrations to use.

Best practices

• In most cases Illumina recommends combining custom Read 1 and Read 2 primers in one well, and custom Index 1 and Index 2 primers in the other well. NextSeq 1000/2000 Custom Primers Guide and NextSeq 1000/2000 Control Software allow any combination of sequencing primers in one well however, keep in mind the following (relevant to most library types):

  • Read 1 and i7 index (index 1) are sequenced on the same strand and Read 2 and i5 index (index 2) are also sequenced on the same strand.

  • Read 1 and i5 index (index 2) and Read 2 and i7 index (index 1) are the reverse-complement of each other and will bind to each other.

  • For reference, see figure 1 below.

  

Figure 1: The NextSeq 1000/2000 Reverse Complement Workflow Index Sequencing Scheme shows the binding region of read and index primers

• Combine no more than 2 custom primers per well. See Figured 2 below.

  

Figure 2: Reagent Cartridge layout detailing the location of custom primer wells (Well 1 and Well 2).

PhiX Considerations

PhiX requires the Illumina sequencing primers for proper priming and sequencing. When custom primers are selected, the standard Illumina sequencing primers are not used for the read or index priming during the sequencing run. If Illumina primers are not used for Read 1 or Read 2, the optional PhiX control is not primed and will not be sequenced.

• If the library pool includes optional PhiX spike-in or requires Illumina primers combined with custom sequencing primers, an additional NextSeq 1000/2000 primer kit must be purchased and added to the custom primer wells.

Kit name	Catalog#
Standard SBS NextSeq 1000/2000 Read & Index Primers	20046115
Standard SBS NextSeq 1000/2000 Index Primer Kit	20046116
Standard SBS NextSeq 1000/2000 Read Primer Kit	20046117
XLEAP-SBS NextSeq 1000/2000 Read & Index Primer	20112856
XLEAP-SBS NextSeq 1000/2000 Index Primer Kit	20112858
XLEAP-SBS NextSeq 1000/2000 Read Primer Kit	20112859

How to designate Custom Primers in Run Setup

Custom primers are compatible with Cloud, Hybrid, Local, and Standalone run modes. Indicate in the control software run setup page if a read requires a custom primer. Define which well (see Figure 3) for each read.

• For Cloud, Hybrid, or Local modes, custom primer selection takes place in the NextSeq 1000/2000 Control Software on the Review screen and is not setup in BaseSpace Run Planning or on the Sample Sheet.

• For Standalone Mode, Custom Primers are selected on the Run Setup screen.

  

Figure 3: Run Setup overview for custom primer selection.

Custom primer selections are indicated in the runparameters.xml file.

Table 1: Comparison of RunParameters.xml entries with and without custom primer usage.

Custom Primer in Read 1 alone	No Custom Primer Used

Custom primer recipes are available for the v2 P2 cartridge. These can be found on the NextSeq 1000/2000 Sequencing System Product Files page under NextSeq 1000/2000 P2 (v2) Reagents Kit Custom Recipes for Custom Primers.

For any feedback or questions regarding this article (Illumina Knowledge Article #2431), contact Illumina Technical Support [e-mail].